Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: A. Illustration of OPN isoform primary domain structure. Each block corresponds to an exon (numbered). OPNa is full length (top), OPNb lacks exon 5 (middle), and OPNc lacks exon 4 (bottom). Expanded amino acid sequences of exons 4 and 5, absent in OPNc and OPNb, respectively, are included. B. OPN primers were used to measure OPNa (277bp), OPNb (235bp), and OPNc (196bp) mRNA levels in tissue samples from non-ischemic tissues and tissues from PAD patients with critical limb ischemia by RT-PCR (n=3–5). Isoform plasmid DNA controls and beta actin for loading are both shown. C. To investigate if OPN isoforms differentially affect collateral vessel formation in vivo , the hindlimb ischemia was performed on the following groups: WT, OPN −/− , or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. Perfusion was measured by Laser Doppler perfusion imaging (LDPI). Representative LDPI images 14 days post-HLI are shown. D. Ischemic limb (IL) perfusion was quantified and normalized to the contralateral non-ischemic limb (NIL) and compared across groups. * p<0.05, † p<0.001 vs. GFP; d14, n=6. E. To determine if the OPN isoform effects on perfusion translate to increased functional limb use, animals were given free access to a running wheel (d7 post-HLI) and allowed to run for 7 days. Total distance run (meters) by was plotted for all groups as a measure of limb function. *p<0.05, † p<0.001 vs. GFP; n=9–10.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, In Vivo, Imaging, Functional Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: To determine if OPN isoforms differentially affect arteriogenesis, tissue sections from animals 14 days post-HLI treated (trx) with lentivirus (LV) to overexpress OPN isoform a, b, or c were stained with α smooth muscle actin (α-SMA). α-SMA positive vessel numbers and sizes were quantified as a readout for arteriogenesis. A. The number of α-SMA positive vessels was counted across treatment groups and plotted (p = ns). B. α-SMA positive vessel sizes were measured in WT or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. The number of vessels measured within the arteriole (10 – 200 μm 2 ), small artery (200 – 700 μm 2 ) and large artery (1000 – 2500 μm 2 ) size ranges were compared across all animal groups. Data are expressed as % change compared to +LV-GFP (control). *p<0.05 vs. OPNa, † p<0.001 vs. OPNb; n=8–10. C. Representative histology images from 14 days post-HLI stained with α-SMA are shown. α-SMA stain is red and counterstain is violet. Scale bars = 500 μm.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Staining